JP5582476B2 - Marine plankton-derived photoprotein - Google Patents

Description

  The present invention relates to a novel photoprotein (luciferase) derived from marine plankton and a gene group thereof, an ancestor photoprotein having photoprotein activity estimated from them, and a gene encoding the ancestor photoprotein.

  Luciferase (FLuc) derived from the North American firefly Photinus pyralis (FLuc), or a variant protein thereof, can be recombinantly expressed in heterologous cells, for example, various mammalian cells, and the resulting set The modified protein exhibits luminescent properties by decomposing the luminescent substrate D-luciferin in the host cell. Similarly, Renilla reniformis-derived luciferase (RLuc: Renilla luciferase) can be expressed in various organisms including animal cells, and has a luminescent activity of degrading the luminescent substrate coelenterazine. The amount of luminescence obtained by these luciferases and their mutants is excellent in quantification, and mainly measures RNA transcription activity of animal cell promoters (gene expression control regions) in the fields of biochemistry, cell biology, and medicine. Widely used as a reporter protein for promoter assays (Non-patent Document 1).

  On the other hand, in addition to the luciferases derived from P. pyralis and R. reniformis, secretory luciferases have been cloned from Vargula hilgendorfii (Non-patent Document 2), and the same arthropoda (Arthropoda) copepod subclass. A secreted luciferase has also been cloned from Gaussia princeps (Copepoda) (Non-patent Document 3). Because the luciferase from G. princeps of these copepoda (Copepoda) specifically degrades a luminescent substrate structurally different from V. hilgendorfii and North American firefly (P. pyralis), Biological evolution has been reported to have different origins. The luminescent substrate (luciferin) has been identified to be coelenterazine as is R. reniformis-derived luciferase.

Other luciferases derived from the same Copepoda Metridia longa (Non-patent Document 4), luciferases derived from Metridia pacifica (Non-patent Document 5), which we previously cloned, (Aequorea victoria) -derived aequorin (Non-patent document 1), decapoda (Hydro shrimp) (Oplophorus gracilirostris) -derived luciferase (Non-patent document 6), etc., the coelenterazine as the luminescent substrate (luciferin), It is reported that it can be used.

  Luciferase (FLuc) from North American firefly (P. pyralis) and R. reniformis luciferase (RLuc) are widely used as in vivo luminescent reporter proteins that can be stably expressed in animal cells. Is a non-secretory luciferase. Therefore, in order to perform a promoter assay, it is necessary to disrupt luciferase activity by disrupting host animal cells and the like. On the other hand, luciferases derived from marine organisms, especially the zooplankton species G. princeps, Metridia longa, and Metridia pacifica, have the same luminescent substrate (coelenterazine) as R. reniformis. ) Oxidatively decomposes and emits light, but unlike R. reniformis, it is a secreted luciferase. Therefore, the reporter luciferase protein is rapidly secreted outside the host cell, and the luciferase activity in the medium may be monitored, and there is no need for cell disruption. As a result, continuous monitoring and time course experiments using the same cells can be easily performed. Cypridina (V. hilgendorfii) luciferase is also secreted, but its luminescent substrate, Cypridina luciferin, has a completely different structure from coelenterazine. In addition, luciferase derived from G. princeps and M. pacifica has been reported to show remarkable heat resistance, but has a low resistance to organic solvents, etc. There is room for further improvement (Non-Patent Document 5, Non-Patent Document 7).

  Therefore, there has been a long-awaited search and development of a novel luciferase that can be expressed in host animal cells and has higher brightness and stability.

  When the luciferase protein is used as an in vivo luminescent reporter protein that can be expressed in the host cell, the luciferase protein is translated in the host cell using a specific promoter. The translated luciferase protein is folded into an appropriate structure and exhibits its luminescent activity. In general, fluorescent marker proteins have been used to observe the function and localization of a specific protein. That is, it is expressed in a form in which the amino acid sequence of the fluorescent marker protein is fused to the N-terminus or C-terminus of a specific protein, and the host cell is irradiated with excitation light to detect fluorescence obtained from the fluorescent marker protein. For example, Dr. Shimomura's research and GFP, which has received particular attention because they were awarded the Nobel Prize, have often been used. Recently, however, a system for detecting intracellular signals by fusing a luciferase protein to a functional peptide or protein has also been developed (Non-patent Document 8). In such a detection system, the molecular weight of the luciferase to be fused greatly affects the function of the fusion protein. The luciferase (FLuc) derived from North American firefly (P. pyralis), which has been widely used as a luciferase protein, is 61 kDa, the luciferase (RLuc) derived from R. reniformis is 36 kDa, and derived from V. hilgendorfii The luciferase of 62 kDa has a relatively high molecular weight as a fusion protein, and it has been desired to provide a novel luminescent reporter protein having a smaller molecular weight and higher luminance. The molecular weights of the luciferases from the zooplankton species G. princeps, M. longa and M. pacifica are 19.9 kDa, 23.8 kDa and 22.7 kDa, respectively. Was less than 30 kDa.

Bioluminescence, chemical principles and methods, Osamu Shimomura, World Scientific Publishing Co. Ltd, 2006 Thompson, EM, Nagata, S, and Tsuji, FI.Cloning and expression of cDNA for the luciferase from the marine ostracod Vargula hilgendorfii. Proc. Natl. Acad. Sci. USA, 86, 6567-6571, 1989 Verhaegen, M, and Christopoulos, TK. Recombinant Gaussia luciferase. Overexpression, purification, and analytical application of a bioluminescent reporter for DNA hybridization. Anal. Chem., 74, 4378-4385, 2002 Markova, SV, Golz, S., Frank, LA, Kalthof, B. and Vysotski, ES.Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa.J. Biol. Chem., 279, 3212-3217, 2004 Takenaka, Y, Masuda, H, Yamaguchi, A, Nishikawa, S, Shigeri, Y, Yoshida, Y, and Mizuno, H. Two forms of secreted and thermostable luciferase from the marine copepod crustacean, Metridia pacifica. Gene, 425, 28 -35, 2008 Inouye, S, and Sasaki, S. Overexpression, purification and characterization of the catalytic component of Oplophorus luciferase in the deep-sea shrimp, Oplophorus gracilirostris. Protein Expr. Purif., 56, 261-268, 2007 Rathnayaka, T, Tawa, M, Sohya, S, Yohda, M, and Kuroda, Y. Biophysical characterization of highly active recombinant Gaussia luciferase expressed in Escherichia coli. Biochim. Biophys. Acta, 1804, 1902-1907, 2010 Kim, S, Sato, M, and Tao, H. Genetically encoded bioluminescent indicators for stress hormones. Anal. Chem., 81, 3760-3768, 2009

  An object of the present invention is to provide a novel photoprotein and a gene encoding the photoprotein.

  Therefore, in order to solve the above-mentioned problems, marine plankton having luminescent ability was collected in the sea near Japan, specifically off the coast of Hakodate Bay, Oyashio region, and the photoprotein (luciferase) was comprehensively cloned. Based on the bioinformatics, the ancestor gene of luciferase was predicted, the minimum functional region essential for luciferase activity was identified from this sequence, and a luciferase with a molecular weight of several tens of kDa that retains the luminescence function was developed. did.

The present invention provides the following novel photoproteins and genes encoding them.
Item 1. A photoprotein comprising the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 24, or a modified amino acid sequence in which one or more amino acids are substituted, added, deleted or inserted.
Item 2. Item 2. The photoprotein according to Item 1, having any one of the amino acid sequences of SEQ ID NOS: 1 to 21.
Item 3. Item 3. A DNA encoding the photoprotein according to Item 1 or 2, or a complementary strand thereof.

  According to the present invention, the ancestral gene and amino acid sequence of the photoprotein could be identified.

  The discovery of such ancestral genes is expected to develop modified photoproteins with better properties.

The alignment of the amino acid sequences of the luciferases derived from the Metridinidae family and the Lucicutiidae family and the modified ancestor luciferase aCopLuc43mut (SEQ ID NO: 22) and the ancestor photoprotein aCopLuc43 (SEQ ID NO: 23) before modification is shown. The alignment of the amino acid sequence of luciferase derived from the Heterorhabdidae family and the ancestor photoprotein aCopLuc48 (SEQ ID NO: 24) deduced therefrom is shown. FIG. 2 shows (a) a fluorescence micrograph under ultraviolet irradiation and (b) a micrograph under white light of the luminescent plankton Metridia okhotensis. Fig. 2 shows the activity of copepod-derived luciferase secreted and expressed in the culture medium by cultured human cells (HEK293). In Example 2, the luciferase activity of the ancestor genes (aCopLuc43mut and aCopLuc48) expressed by E. coli is shown. In Example 3, the luciferase activity of the ancestor genes (aCopLuc43mut and aCopLuc48) expressed by cultured human cells (HEK293) is shown.

  One feature of the present invention is to identify the gene sequence and amino acid sequence of luciferase derived from Metridinidae, Lucicutiidae or Heterorhabdidae plankton in marine plankton-derived photoproteins, thereby determining the ancestral gene and amino acid sequence. is there. The amino acid sequence of the ancestral photoprotein of Metridinidae family plankton and Lucicutiidae family plankton is shown in SEQ ID NO: 22, and the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 46. The amino acid sequence of the ancestral photoprotein of Heterorhabdidae family plankton is shown in SEQ ID NO: 24, and the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 48.

  The amino acid sequence set forth in SEQ ID NO: 22 is the amino acid sequence set forth in SEQ ID NO: 23 in which Cys → Asp at position 91 is replaced with Ser at position 92.

  The ancestral photoprotein of SEQ ID NO: 23 obtained using the photoprotein derived from the marine plankton of the Metridinidae family and Lucicutiidae family found by the present inventors (SEQ ID NO: 1 to 15) did not have luminescent activity. It was confirmed that the modified ancestor photoprotein having the amino acid sequence set forth in SEQ ID NO: 22 in which the amino acids at positions # 92 and # 92 were substituted as described above had luminescence activity.

  In the case of Heterorhabdidae family plankton, it was confirmed that the ancestor photoprotein (SEQ ID NO: 24) obtained using the amino acid sequences of SEQ ID NOs: 16 to 21 found by the present inventors has a luminescent activity.

  The ancestral photoproteins shown in SEQ ID NOs: 22 and 24 have been revealed for the first time by the present invention.

  In the present invention, amino acid substitutions are possible within the ranges shown in FIG. 1 (excluding aCopLuc43 of SEQ ID NO: 23) and FIG. 2 in the sequences of SEQ ID NOS: 22 and 24.

  For example, in FIG. 1, the N-terminal amino acid is M in the aCopLuc43mut of SEQ ID NO: 22, but this amino acid can be substituted with H, L, and G that can be confirmed with other photoproteins in FIG. Similarly, the fourth amino acid K of SEQ ID NO: 22 from the N-terminus can be substituted with Q or L. The tenth amino acid V of SEQ ID NO: 22 from the N-terminus cannot be substituted because all other photoproteins are V. Similarly, the 11th amino acid L cannot be substituted. Regarding the amino acid sequence of FIG. 2, the same amino acid substitution is possible in the amino acid sequence of SEQ ID NO: 24.

  Furthermore, other amino acids shown in FIGS. 1 and 2 can be added to the N-terminal or C-terminal side of the amino acid sequences of SEQ ID NOS: 22 and 24.

  In the present invention, the modified amino acid sequence in which one or more amino acids are substituted, added, deleted or inserted in the amino acid sequences of SEQ ID NOs: 22 and 24 includes the above amino acid substitutions or additions. Here, the one or more amino acids to be substituted, added, deleted or inserted are 150 or less, 145 or less, 140 or less, 135 or less, 130 or less, 125 or less, 120 or less, 115 110 or less, 105 or less, 100 or less, 95 or less, 90 or less, 85 or less, 80 or less, 75 or less, 70 or less, 65 or less, 60 or less, 55 or less, 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, 25 or less, 20 or less (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acid substitutions, additions, deletions or insertions. For example, MoLuc1 shown in FIG. 1 is a modified ancestor photoprotein having the number shown in FIG. 1 and a total of 80 amino acids of SEQ ID NO: 22, from No. 1 to 48, 57 to 85, 146, 148, and 226 In addition, 87 (P / G), 94 (A / E), 97 (I / K), 104 (F / K), 118 (K / H), 127 ( E / K), 128 (Y / F), 135 (D / S), 137 (G / E), 141 (K / D), 143 (G / A), 145 (A / G), 149 (V / I), 151 (A / P), 159 (S / P), 163 (E / D), 165 (G / A), 178 (A / V) 183 (G / R), 192 (K / Q), 195 (A / D), 196 (L / Q), 203 (D / T), 206 (A / T), 207 30 (S / T), 210 (D / S), 214 (R / K), 217 (H / D), 218 (N / T), 224 (G / D) Of amino acids are substituted. Therefore, in the case of MoLuc1, 110 amino acids are substituted, added, deleted or inserted into the amino acid sequence of SEQ ID NO: 22, and such amino acid substitution, addition, deletion or insertion is performed according to the present invention. Is within the range. In addition, substitution, addition, deletion, and insertion of amino acids are preferably within the range shown in FIGS. 1 and 2, and a maximum of 110 amino acids compared to MoLuc1 and the ancestor photoprotein (aCopLuc43Mut) of SEQ ID NO: 22. These amino acids can be substituted, added, deleted or inserted, but 1 to 109 amino acids may be appropriately selected and substituted, added, deleted or inserted. The above description is explained by taking MoLuc1 as an example, but MoLuc2, McLuc1, McLuc2, MaLuc1, MaLuc2, PsLuc1, PsLuc2, PaLuc1, PaLuc2, PxLuc1-7, PxLuc1-8, PxLuc2, LoLuc1-1, LoLuc1-3 Similarly, amino acid substitutions, additions, deletions or insertions are possible within the range shown in FIG. Further, HtLuc1-1-2, HtLuc1-1-3, HtLuc1-2-2, HtLuc2, HmLuc1, and HmLuc2 can similarly be substituted, added, deleted, or inserted within the range shown in FIG. is there.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail according to an Example, it cannot be overemphasized that this invention is not limited to these Examples.

Example 1: Luminescent plankton-derived novel luciferase
1. Amino acid sequence and base sequence The luminescent plankton was searched from the zooplankton inhabiting the ocean. Specifically, in collaboration with Atsushi Yamaguchi (Visiting Researcher, AIST) at the Graduate School of Fisheries Sciences, Hokkaido University, we boarded an attached training ship, Oshoro Maru, in the ocean collected off the coast of Hakodate Bay and in the Oyashio region. A number of luminescent plankton were collected in the process of classification of zooplankton. Furthermore, among these luminescent planktons, taxonomically selected zooplankton belonging to Arthropoda and Copepoda that express secretory expression photoproteins outside the body. did. Attempts were made to classify Copepoda that expresses secreted photoproteins found in this zooplankton collection, and the plankton of the Metridinidae family, Lucicutiidae family or Heterorhabdidae family belonging to the superfamily Calanoida, Augaptiloidea Identification was performed. Furthermore, we identified the nucleotide sequence of the gene encoding it and determined the deduced amino acid sequence of the luciferase protein from Metridinidae, Lucicutiidae or Heterorhabdidae plankton. First, total RNA was extracted from Metridinidae family, Lucicutiidae family or Heterorhabdidae family plankton, the contained mRNA was purified, and the corresponding cDNA was synthesized according to a standard method using reverse transcriptase. A partial sequence was amplified from cDNA by PCR using the luciferase gene sequence derived from Metridinidae family plankton reported so far. As a result, a partial sequence of a luciferase gene of about 250 bases was obtained. Next, based on the nucleotide sequence, the full-length cDNA was amplified and the nucleotide sequence was analyzed, and the sequence of the luciferase protein derived from the intended Metridinidae, Lucicutiidae, or Heterorhabdidae plankton was elucidated. Align the full-length amino acid sequence of the luciferase protein from this Metridinidae, Lucicutiidae or Heterorhabdidae plankton, and use the computer program (MEGA5) to predict the ancestor luciferase protein sequence with molecular weights of 15 kDa and 13.8 kDa The gene sequence to be encoded was artificially synthesized and expressed in Escherichia coli and mammalian cultured cells to confirm its luciferase function.

  It is inferred from the amino acid sequences of the photoproteins derived from the zooplankton belonging to the family Calanoida, Augaptiloidea, Metridinidae, Lucicutiidae, Heterorhabdidae, and the zooplankton belonging to the copepod Metridinidae and Lucicutiidae according to the present invention. Its ancestor luciferase protein (aCopLuc43), its variant (aCopLuc43mut), the name of the ancestor luciferase protein (aCopLuc48) deduced from the amino acid sequence of the photoprotein derived from the zooplankton belonging to the copepod Heterorhabdidae, the full-length amino acid sequence, base The correspondence relationship of the sequence numbers is shown below.

2. Experimental method With the expression of the protein in the plankton body of the copepod Metridinidae, Lucicutiidae or Heterorhabdidae, we attempted to select the gene of the luciferase from the various remaining mRNAs. Specifically, a cDNA library was prepared from mRNA, and a partial sequence and a full-length sequence of the luciferase gene were obtained from this cDNA library by PCR.

  Hereinafter, the luciferase derived from copepods of the present invention will be described in more detail.

Collection and identification of copepod luminescent plankton First, the animal luminescent plankton, which is the origin of the luciferase of the present invention, was found in the deep water of Oyashio region collected from the depth of 50 to 1000 m off the coast of Hakodate, Calanoida, Augaptiloidea It is a plankton belonging to the superfamily. As shown in FIG. 3 (a), a typical luminescent plankton, Metridia okhotensis, has a secretory gland containing a luminescent substrate that looks blue-green when observed with a fluorescence microscope under ultraviolet irradiation. When the microscope is observed under white light, the plankton body of Metridia okhotensis is observed from transparent to slightly white as shown in FIG. 3 (b). The plankton on which the luciferase gene was cloned in the present invention belongs to Metridinidae (Metridinidae), Metridia okhotensis, Metridia curticauda, Metridia asymmetrica, Pleuromamma abdominalis, Pleuromamma scutullata, Pleuromamma xiphias, Lucicutiidae. ovaliformis and Heterorhabdidae (Heterohabdidae) belong to Heterorhabdus tanneri and Heterostylites major. The detailed taxonomic identification of these zooplankton includes Metazoa (Animal kingdom), Arthropoda (Arthropoda), Crustacea (Crustacea), Maxillopoda (Copepoda), Copepoda (Copepoda), Neocopepoda ( (Copepoda), Gymnoplea (Antipoda), Calanoida (Calanus), Metridinidae (Metridinidae) or Lucicutiidae (Helicidae) or Heterorhabdidae (Heterohabdidae).

  The present inventors show luminescence when a plankton belonging to the Metridinidae family, Lucicutiidae family or Heterorhabdidae family gives a physical stimulus, and luminescence is observed when the crude extract is mixed with a luminescent substrate coelenterazine. As a result of detailed examination, it was concluded that it was not caused by the infestation and adhesion of bacteria producing luciferase, but due to luciferase derived from the plankton itself of this family of Metridinidae, Lucicutiidae, or Heterorhabdidae. Therefore, the plankton was collected, and it was considered to proceed with the isolation of the luciferase protein. However, the amount of plankton obtained was not sufficient, and it was determined that it was difficult to recover a sufficient amount of protein for the amino acid sequence analysis. It was. Therefore, based on the amino acid sequence of luciferase derived from Metridia longa of the closely related Metridia genus (GenBank accession number, AY364164; literature 4) and luciferase derived from Gaussia princeps (GenBank accession number, AY015993; literature 3) belonging to the genus Gaussia, A method was used in which a degenerate primer encoding a part of an amino acid sequence having high homology was prepared and the gene of the luciferase was cloned from cDNA by PCR.

Purification and cDNA synthesis of copepod luminescent plankton RNA
Extraction of total RNA from Metridinidae, Lucicutiidae or Heterorhabdidae plankton using a commercially available RNA extraction reagent; RNeasy Mini kit (Qiagen), followed by a commercially available purification kit; Oligotex-dT30 <SUPER> mRNA Purification kit Poly (A) + mRNA was purified using (Takara Bio). Furthermore, cDNAs for 5 'end amplification and 3' end amplification were synthesized from purified mRNA using a commercially available cDNA preparation kit: SMART RACE cDNA amplification kit (Takara Bio), respectively. .

Obtaining copepod luciferase cDNA fragment by PCR reaction
In the gene cloning process encoding luciferase derived from plankton from the family Metridinidae, Lucicutiidae or Heterorhabdidae, the two upstream mixed primers and two downstream mixed primers were combined using cDNA prepared from mRNA as a template. The PCR reaction was performed for a total of 4 types of PCR primer pairs.

  Specifically, the base sequences of two types of upstream mixed primers and two types of downstream mixed primers shown below were selected.

Upstream mixed primer
White luc UP1 (26 mer: 16 mixed primers)
5'-GGC TGC ACY AGG GGA TGY CTK ATM TC-3 '
(Y = T, C; K = G, T; M = A, C)
White luc UP2 (26 mer: 16 mixed primers)
5'-GCT ATT GTT GAY ATY CCY GAR AT-3 '
(Y = T, C; R = G, A)

Downstream mixed primer
White luc LP2 (26 mer: 16 mixed primers)
5'-TC AAG TTG WTC AAT RAA YTG YTC CAT-3 '
(W = A, T; R = G, A; Y = T, C)
White luc LP1 (23 mer: 12 mixed primers)
5'-AC ATT GGC AAG ACC YTT VAG RCA-3 '
(Y = T, C; V = A, G, C; R = G, A)

  A PCR amplification product is prepared from the 3'-Ready cDNA prepared using mRNA derived from Metridinidae family, Lucicutiidae family or Heterorhabdidae family plankton as a template according to the conventional method using the 4 pairs of primer pairs shown above.

  To the recovered DNA solution (PCR amplification product), 2 μL of 10 × Loading dye solution was added, and then applied to 1.6% TAE agarose gel, and 15 μL of each lane DNA solution was electrophoresed. A band of the desired molecular weight is cut out from the gel, and the gel piece is placed in a microtube column included in a Montage gel extraction kit (Millipore) and centrifuged at 7,000 rpm for 10 minutes. The DNA solution extracted through the membrane is further purified using a MinElute PCR purification kit (Qiagen). 5 volumes of buffer PB was added per volume of DNA solution, vortexed, and transferred to a MinElute column. Centrifuge for 30 seconds to adsorb the DNA to the column. The column is then washed with 0.7 mL of buffer PE and the buffer PE is completely removed from the column by centrifugation (15,000 rpm) for an additional minute. Thereafter, 10 μL of elution buffer EB is added, and the mixture is allowed to stand at room temperature for 1 minute. Finally, the mixture is centrifuged (15,000 rpm) for 1 minute, and the DNA eluted from the column is collected in a 1.5 mL microtube.

  The purified PCR reaction product is inserted into the cloning vector pCR2.1-TOPO (Invitrogen). Ligation is performed by adding 0.5 μL of Salt solution and 0.5 μL of the vector solution attached to the vector to 2 μL of the PCR reaction product, and incubating at room temperature for 5 to 15 minutes. DNA contained in this reaction solution is introduced into a Chemical competent cell DH5alpha strain, and a transformant is selected. The plasmid vector was introduced into host E. coli according to the following procedure. The DH5alpha strain used for host Escherichia coli thaws the cryopreserved competent cell at ice temperature. Add 3 μL of the PCR product insertion plasmid vector solution to 50 μL of the thawed host E. coli DH5alpha strain suspension. After being kept on ice for 10 minutes, it was heated at 42 ° C. for 30 seconds and returned to ice. Next, 100 μL of SOC medium is added, followed by shaking culture at 37 ° C. for 10 minutes. Thereafter, a transformant strain contained in the culture solution is selected using a selection marker derived from the cloning vector pCR2.1-TOPO. Colonies appearing on LB plates containing 50 μg / mL antibiotic carbenicillin are analyzed to select E. coli having a plasmid vector carrying the PCR product.

Plasmid vector holding the PCR product is purified and purified. From the selected clone, the introduced plasmid vector is purified by the following procedure.

  Colonies (clones) of the host E. coli DH5alpha strain holding the plasmid vector into which the PCR product was inserted were suspended in 2 mL of LB / carbenicillin liquid medium and cultured at 37 ° C. for 16 hours. The culture solution was centrifuged (5,000 × g) for 10 minutes to separate cells.

  From the sorted cells, the plasmid is separated and purified using a commercially available plasmid purification kit: Qiagen Plasmid purification kit (Qiagen). To the sorted cells, add 0.25 mL of P1 solution attached to the purification kit, and vortex to disperse well. To this cell dispersion, add 0.25 mL of the attached P2 solution, mix, and let stand at room temperature (20 ° C.) for 5 minutes. After lysis treatment, add 0.35mL of the attached N3 solution and mix. Subsequently, it is centrifuged (15,000 rpm) at 4 ° C. for 15 minutes, and the supernatant containing the plasmid DNA is separated and collected. The supernatant is applied to the QIAprep mini column of the same purification kit. Centrifuge (15,000 rpm) at 4 ° C to remove the liquid layer. Add 0.5 mL of buffer PB to wash the column, then add 0.75 mL of buffer PE and wash again. Finally, centrifuge (15,000 rpm) for 1 minute at 4 ° C to completely remove the washing solution. The plasmid DNA adsorbed on the QIAprep mini column of the purification kit is eluted and collected with 30 μL of buffer EB. Of 30 μL of the purified plasmid-containing solution, 1 μL was taken, 99 μL of distilled water was added, and the concentration of DNA was determined by measuring the absorbance at 260 nm as a 100-fold diluted solution. The concentration of the plasmid vector was adjusted so as to be 250 ng / μL.

Analysis of nucleotide sequence of cDNA fragment (PCR product) in selected clone A nucleic acid chain extension reaction product for nucleotide sequence analysis is prepared from the cDNA fragment (PCR product) inserted in the plasmid vector by the following procedure.

  Collected from selected clones, then sampled for nucleotide sequence analysis using this purified plasmid vector as a template using a commercially available DNA sample preparation kit for sequencing: BigDye Terminator Cycle Sequencing Ready Reaction Kit ver. 1.1 (manufactured by Life Technologies Japan) Is used as a sequence primer using two primers; M13 sense M4 and M13 reverse, to prepare a sequence reaction product of the region containing the cDNA fragment inserted in the plasmid vector. The prepared sample for base sequence analysis is purified by the following procedure.

  Transfer the prepared sample solution from each reaction tube to a separate 0.5 mL tube. Prepare a mixture of 0.5 M 3M aqueous sodium acetate solution and 12.5 μL of 95% ethanol per 5 μL sample solution separately in a 1.5 mL tube. The sample solution collected in advance is put into the 1.5 mL tube. After mixing uniformly, the mixture is allowed to stand for 10 minutes under ice cooling to precipitate the contained DNA fragment in ethanol. Centrifuge (15,000 rpm) for 20 minutes to deposit the precipitated DNA fragments, and remove the supernatant. 125 μL of 70% ethanol is then added to rinse the precipitated DNA. Again, centrifuge (15,000 rpm) for 5 minutes and aspirate the supernatant. Dry the pellet of the remaining precipitated DNA fragments.

  The purified sample DNA fragment for analysis is redissolved in 15 μL formamide. Vortex to mix, then centrifuge to collect liquid. Heat at 95 ° C for 2 minutes, separate into single-stranded DNA, and cool on ice. Thereafter, the sample DNA fragment for analysis is subjected to nucleotide sequence analysis by applying it to a commercially available sequence device: ABI PRISM 3100 Genetic Analyzer (manufactured by Life Technologies Japan).

  The sequence analysis result of the sense strand and the sequence analysis result of the antisense strand are combined to determine the base sequence of the inserted cDNA fragment (PCR reaction product).

  The base sequence and amino acid sequence of each luciferase derived from copepods are shown in Table 1 and Sequence Listing.

Introduction of the expression vector for the expression of human cells into the HEK293 cells of the copepod-derived luciferase protein into the mammalian cell expression vector pcDNA3.2 V5-His TOPO (manufactured by Invitrogen) code of the copepod-derived luciferase gene obtained above Insert an area. Confirm by sequencing that each gene is inserted into the cloning site of plasmid pcDNA3.2 V5-His TOPO. Of the prepared mammalian cultured cell expression vectors, MoLuc1, MoLuc2, MaLuc1, PsLuc1, PaLuc1, PaLuc2, PxLuc1-7, PxLuc1-8, LoLuc1-1, LoLuc1-3 were gene-expressed as representatives. The purified human cell expression vector was transfected into HEK293 cells using Lipofectamine 2000 (Invitrogen). HEK293 cells of the host are obtained by culturing to 95% confluent state on a 100 mm dish using 10 mL serum-supplemented medium (MEM + 10% FBS + Penicillin / streptomycin). First, 24 μL of the plasmid solution (DNA concentration: 1 μg / μL) is suspended in 1.5 mL of serum-free Opti-MEM medium, and 60 μL of Lipofectamine 2000 is similarly suspended in 1.5 mL of serum-free Opti-MEM medium. Prepare what you did. Mix the two solutions and incubate at room temperature for 20 minutes. Add the entire volume of the mixture to the cell culture medium on a 100 mm dish, and gently stir to make it uniform. After the transfection treatment, HEK293 cells are incubated for 48 hours or more at 37 ° C. under 5% CO ₂ to express the transgene.

  All 10 mL of the culture supernatant was transferred to a new 15 mL centrifuge tube and centrifuged at 1000 rpm for 2 minutes, and the centrifuged supernatant was used as a medium fraction. On the other hand, 10 mL of fresh medium was added to the cells remaining on the dish, and the cells were removed using a cell scraper, and then all of the cells were collected in a 15 mL centrifuge tube. The suspension was sonicated on ice for 10 seconds and centrifuged at 15,000 rpm for 5 minutes. The supernatant was collected as an intracellular fraction, and the luminescence activity was measured simultaneously with the medium fraction. It was observed that the protein was secreted extracellularly and accumulated in the medium (FIG. 4). The secretion efficiency at that time is shown in Table 2. The method for measuring luciferase activity was described in “Measurement of copepod-derived luciferase activity” described later. As described above, even in human-derived HEK293 cells, translation into a pre-protein type recombinant having a full-length amino acid sequence is performed based on the coding gene of copepod-derived luciferase protein. Thereafter, it is clearly shown that it is secreted out of the cell as a mature active protein using the signal peptide portion present at its N-terminus.

Example 2: Prediction of ancestor gene, expression in E. coli, and confirmation of activity
Prediction of ancestor gene of copepod luciferase
The amino acid sequence of luciferase from Metridinidae, Lucicutiidae and Heterorhabdidae plankton was analyzed with MEGA5 software for molecular evolution and phylogenetic analysis of protein sequence data for each family. First, multiple sequence alignment was performed using ClustalW based on the amino acid sequence. Based on this multiple sequence alignment data, the phylogenetic tree was reconstructed by the Maximum likelihood method, and the ancestral gene sequence estimation function of MEGA5 was used to estimate the ancestral gene sequence. Specifically, aCopLuc43 consisting of 137 amino acids as the amino acid sequence of its ancestral gene deduced from the amino acid sequences of 15 luciferases derived from the plankton of the Metridinidae and Lucicutiidae families, and 6 amino acids of the 6 luciferases derived from the Heterorhabdidae family plankton The aCopLuc48 consisting of 132 amino acids was determined as the amino acid sequence of the ancestral gene deduced from the sequence. The reason why the common ancestral genes of the Metridinidae and Lucicutiidae families were estimated was that the amino acid sequences of the luciferases found in these two families were higher in homology than those from the Heterorhabdidae family. The amino acid sequence of this aCopLuc43 was aligned with 15 luciferases derived from the cloned plankton of the Metridinidae and Lucicutiidae families (Fig. 1), and the amino acid residue Cys strongly conserved in the amino acid sequences of the 15 luciferases. -X (3) -Cys-Leu-X (2) -Leu-X (4) -Cys-X (8) -Pro-X-Arg-Cys (X, amino acid) Then, after artificially synthesizing the base sequence encoding the amino acid sequence of aCopLuc43, mutations were introduced into the sites (91st and 92nd positions of SEQ ID NO: 23), and a clone that matched this rule, aCopLuc43mut (SEQ ID NO: 22) was obtained. Created.

Expression by Escherichia coli of ancestor gene of copepod luciferase
Nucleotide sequences encoding the ancestral genes aCopLuc43, aCopLuc43mut and aCopLuc48 of copepod luciferase obtained by analysis of the amino acid sequence of luciferase from Metridinidae, Lucicutiidae and Heterorhabdidae plankton are optimal for the codon frequency used in E. coli And synthesized using Operon Biotechnology's artificial gene synthesis service. This plasmid vector was subjected to restriction enzyme treatment with NdeI and BamHI, the insert DNA fragment was run on a 1.6% agarose gel, the restriction enzyme fragment of the target molecular weight was confirmed, and the Montage gel extraction kit (manufactured by Millipore) from the gel slice DNA was recovered by the method described above using MinElute PCR purification kit (manufactured by Qiagen).

The purified insert DNA (coding sequence portion) is ligated into E. coli expression plasmid pET-16b (manufactured by Novagen) similarly treated with NdeI and BamHI. Ligation High (Toyobo Co., Ltd.) was used for ligation. After the reaction, the ligation solution is introduced into the Chemical competent cell DH5alpha strain, and the transformant is selected on the LB / carbenicillin plate. An Escherichia coli clone carrying the expression vector pET-16b having the coding region of the ancestor luciferase gene is selected by screening using colony PCR from colonies that have grown on the plate. The selected colony (clone) is cultured, and plasmid vectors are collected and purified using Qiagen Plasmid purification kit (Qiagen). BigDye Terminator Cycle Sequencing Ready Reaction Kit ver. 1.1 (Life Technologies Japan) Thus, the coding sequence portion of the gene inserted into pET-16b and the base sequence of the ligation portion were sequenced by the method described above, and the gene sequence and orientation were confirmed. The plasmid vector whose sequence has been confirmed is introduced into the E. coli expression strain BL21 CodonPlus RIPL (Stratagene). Transformants were cultured overnight on an LB / carbenicillin plate at 37 ° C., and the resulting colonies were inoculated into 10 mL of ZYM5052 medium (containing 50 μg / mL carbenicillin) and cultured at 16 ° C. and 180 rpm for 6 days. . The composition of the ZYM5052 medium is as follows.
(1% NZ-amine, 0.5% Yeast extract, 25 mM Na ₂ HPO ₄ , 25 mM KH ₂ PO ₄ , 50 mM NH ₄ Cl, 5 mM Na ₂ SO ₄ , 2 mM MgSO ₄ , 0.5% glycerol, 0.05% glucose, 0.2% lactose)
After incubation, centrifuge at 7,000 rpm for 10 minutes to precipitate E. coli, remove the supernatant medium, and resuspend in 1 mL of lysis buffer (20 mM Tris-HCl (pH 8.0), 10 mM MgCl ₂ ). It became cloudy. The suspension was sonicated on ice for 10 seconds and centrifuged at 15,000 rpm for 5 minutes. The supernatant was collected as a fraction containing a soluble recombinant protein, and its activity was measured to find luminescence activity (FIG. 5).

Example 3: Expression of ancestral genes in human cells (HEK293) (FIG. 6)
Expression of ancestor gene of copepod luciferase by human cells
The base sequence encoding the amino acid sequences of the ancestor genes aCopLuc43mut and aCopLuc48 of copepod luciferase obtained by analysis of the amino acid sequence of the luciferase from Metridinidae, Lucicutiidae and Heterorhabdidae plankton is expressed in the mammalian cell expression vector pcDNA3.2 V5- Insert into His TOPO. The purified human cell expression vector was transfected and expressed in human cultured cell HEK293, and the luminescence activity of the ancestral gene was measured. HEK293 cells were cultured in a 100 mm dish. After transfection treatment with Lipofectamine 2000 (manufactured by Invitrogen), an expression vector for an ancestor gene of copepod luciferase, the cells were incubated at 37 ° C. under 5% CO _{2 for} 72 hours to express the transgene. As a result, luminescence activity was observed in the intracellular fraction (FIG. 6). The method for measuring luciferase activity was described in “Measurement of copepod-derived luciferase activity” described later.

Measurement of copepod luciferase activity The following method was used to measure the activity of copepod luciferase. 1 μL was taken from a methanol solution of the luminescent substrate coelenterazine prepared to 1 μg / μL, and diluted with 1 mL of substrate dilution buffer (20 mM Tris-HCl, pH 8.0, 50 mM MgCl ₂ ). The diluted luminescent substrate solution was kept on ice. Transfer 5 μL of culture supernatant or E. coli disruption solution from HEK293 cells into which a plasmid vector expressing copepod luciferase has been introduced into a polystyrene test tube and place it in the measurement section of a luminometer LB9506 (Berthold) After the addition of 10 μL of the diluted luminescent substrate, the measurement was started immediately. Luminescence was measured for 10 seconds. When the luminescence was too strong and exceeded the measurement limit, the sample was diluted 10-fold with a sample dilution buffer (20 mM Tris-HCl, pH 8.0, 10 mM MgCl ₂ ) and measured in the same manner. The obtained luminescence value was output to a printer attached to the luminometer, and then the measurement of the next sample was started.

  The luciferase derived from copepods according to the present invention and the gene encoding the protein of its ancestor luciferase can be expressed in the host cell in a culture system using mammalian cells, and The expressed recombinant expression luciferase protein is secreted out of the host cell, or the ancestor luciferase can be used as a reporter protein expressed in the cell.

Claims (3)

Amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 24, or, in the amino acid sequence, 1-6 amino acids are substituted, added, luminescent protein which comprises a deletion or inserted altered amino acid sequence. A photoprotein comprising any one of the amino acid sequences of SEQ ID NOS: 1 to 21. A DNA encoding the photoprotein according to claim 1 or 2, or a complementary strand thereof.

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